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Research Article

A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.

G L Semenza, G L Wang
G L Semenza
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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G L Wang
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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DOI: 10.1128/MCB.12.12.5447
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ABSTRACT

We have identified a 50-nucleotide enhancer from the human erythropoietin gene 3'-flanking sequence which can mediate a sevenfold transcriptional induction in response to hypoxia when cloned 3' to a simian virus 40 promoter-chloramphenicol acetyltransferase reporter gene and transiently expressed in Hep3B cells. Nucleotides (nt) 1 to 33 of this sequence mediate sevenfold induction of reporter gene expression when present in two tandem copies compared with threefold induction when present in a single copy, suggesting that nt 34 to 50 bind a factor which amplifies the induction signal. DNase I footprinting demonstrated binding of a constitutive nuclear factor to nt 26 to 48. Mutagenesis studies revealed that nt 4 to 12 and 19 to 23 are essential for induction, as substitutions at either site eliminated hypoxia-induced expression. Electrophoretic mobility shift assays identified a nuclear factor which bound to a probe spanning nt 1 to 18 but not to a probe containing a mutation which eliminated enhancer function. Factor binding was induced by hypoxia, and its induction was sensitive to cycloheximide treatment. We have thus defined a functionally tripartite, 50-nt hypoxia-inducible enhancer which binds several nuclear factors, one of which is induced by hypoxia via de novo protein synthesis.

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A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.
G L Semenza, G L Wang
Molecular and Cellular Biology Dec 1992, 12 (12) 5447-5454; DOI: 10.1128/MCB.12.12.5447

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A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.
G L Semenza, G L Wang
Molecular and Cellular Biology Dec 1992, 12 (12) 5447-5454; DOI: 10.1128/MCB.12.12.5447
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